Production and characterization of cell-penetrating recombinant botulinum neurotoxin type A

Summary We recently reported that fusion of cell-penetrating peptides (CPPs) to botulinum neurotoxin type A (BoNTA) proteins could improve the efficiency of cellular uptake. Here, we describe steps to produce and evaluate CPP-BoNTA fusion proteins. We present procedures for the expression and purification of recombinant CPP-BoNTA using insect-cell-based baculovirus expression vector system and in vitro characterization of purified proteins. We also detail the analysis of cellular uptake in cell culture and examination of the in vivo performance in mice. For complete details on the use and execution of this protocol, please refer to Wei et al. (2022).1


SUMMARY
We recently reported that fusion of cell-penetrating peptides (CPPs) to botulinum neurotoxin type A (BoNTA) proteins could improve the efficiency of cellular uptake. Here, we describe steps to produce and evaluate CPP-BoNTA fusion proteins. We present procedures for the expression and purification of recombinant CPP-BoNTA using insect-cell-based baculovirus expression vector system and in vitro characterization of purified proteins. We also detail the analysis of cellular uptake in cell culture and examination of the in vivo performance in mice. For complete details on the use and execution of this protocol, please refer to Wei et al. (2022). 1

BEFORE YOU BEGIN
The following protocol outlines the detailed steps for production and characterization of CPP-BoNTA. A complete list of required materials and equipment is provided in the key resources table. Solutions should be prepared in advance according to the recipes and can be stored as indicated.
We will first describe the procedures and strategies used to optimize the expression and purification of recombinant CPP-BoNTA proteins. Then, we describe the experiments for the characterization of purified proteins including the molecular weight and enzymatic activity. Finally, we describe the procedures for analysis of the activity of proteins on cell culture and in mice.

Institutional permissions
All mice used in this experiment are housed in specific pathogen-free (SPF) condition with a 12 h light/ 12 h dark cycle (7 AM-7 PM) and have access to food and water ad libitum at Shanghai Model Organisms Center (Shanghai, China). All mouse experiments are conducted in accordance with the guidelines of the American Association for the Accreditation of Laboratory Animal Care (AAALAC). All animal experimentation is conducted in accordance with the regulations of Animal Care and Use Committee, Shanghai Model Organisms Center, Inc. For researchers who are about to use this protocol in mice, please acquire permissions from your institutions. All mouse husbandry and experiments should be reviewed and approved by the Laboratory Animal Management and Ethics Committee of your institute and should be in strict accordance with good animal practice as defined by the Laboratory Animal Center of your institute.

DNA fragments and expression system
Botulinum neurotoxins (BoNTs) are neurotoxic proteins produced by Clostridium botulinum and related bacterial species. There are at least seven serotypes and forty subtypes of BoNTs. In addition, several BoNT-like proteins have been reported. 2 All types of BoNTs block cholinergic neurotransmission by inhibiting the release of acetylcholine. 3 BoNT type A (BoNTA) is widely used for treating neuromuscular disorders. 4 Naturally occurring BoNTA is composed of a 100 kDa heavy chain (HC) and a 50 kDa light chain (LC). 5 The protein sequence of BoNTA in this study can be found from NCBI.
In the past few decades, many expression systems have been developed, such as Escherichia coli, yeast, mammalian cells and others. Each system has their own advantages. For example, Escherichia coli is known to be affordable and easy to scale up. Mammalian cells can express proteins with posttranslational medications. Importantly, insect cells-baculovirus system has proven efficient for expression of complex proteins. 6 Among the several baculovirus technologies, Bac-to-Bac Baculovirus Expression Vector System (BEVS) provides a rapid and efficient method to generate recombinant baculovirus. The pFastBac1 vector is a suitable vector for both bacterial cloning and protein expression in insect hosts. pFastBac1 contains a polyhedron promoter (P PH ) to drive robust expression of the gene of interest. A Mini Tn7 element is included to permit site-specific transposition of the transgene into the baculovirus genome. 7 We synthesize the target gene from a commercial vendor (GENEWIZ) and use molecular cloning technology to insert the target DNA fragment into the pFast-Bac1 vector.

Cell lines
Prepare Spodoptera frugiperda cells (Sf9) or Trichoplusia ni Hi-5 cells (Hi-5) for baculoviral production and protein expression. Prepare mouse neuroblastoma N2a cells or counterparts for evaluation of the cell-penetrating activity of CPP-BoNTA.

Mice
Prepare 8 to 12-week-old wild-type male or female C57BL/6 mice for intramuscular injection. For each experiment, inject at least five mice per dose. Ni-Wash buffer II.

KEY RESOURCES
Ni-Wash buffer III.
Note: The SEC buffer should be freshly made and kept at 4 C until use.
Note: The SNAPtide assay buffer should be freshly made and kept at 4 C until use.
Cell culture media. This section describes how recombinant CPP-BoNTA proteins are produced. There are three major steps, including plasmid construction (4 days), protein expression (15 days) and protein purification (2 days).

Plasmid construction
Timing: 4 days (for step 1) This section describes how to construct recombinant plasmids encoding CPP-BoNTA proteins. Plasmid construction follows the general molecular cloning procedures and includes 6 sequential steps: PCR reaction, restriction digestion, gel extraction of digestion products, ligation, transformation and bacterial cultivation and DNA purification.
1. PCR Reaction. a. Set up the PCR reaction using an insect cell codon-optimized synthesized BoNTA gene (GENEWIZ).
Note: The forward primers for BoNTA, T-BoNTA and Z-BoNTA (key resources table) all contain N-terminal FLAG and His6 tags ( Figure 1A). The PCR primers introduce 5 0 -XbaI and 3 0 -HindIII restriction sites for cloning into pFastBac1 vector ( Figure 1B).
b. Set up PCR reactions in 8-tube strips.
Note: Master mix (materials and equipment section) should be prepared for 1.23 the number of samples to account for the loss during pipetting process.  c. Set up the PCR reaction as follows: d. Collect the PCR product ( Figure 2A) using MinElute PCR Purification Kit (Qiagen). Elute the DNA using 10 mL of water. Store at À20 C for one week. 2. Restriction digestion.
a. Digest pFastBac1 vector and PCR products with XbaI and HindIII ( Figure 1B). b. Set up digestion in 8-tube strips. Prepare master mix according to the table in the materials and equipment section. c. Incubate the reaction at 37 C for 3 h, and then add calf intestinal alkaline phosphatase (CIP) of 0.5 mL for further incubation for 30 min. 3. Gel Extraction of digested pFastBac1 vector and PCR amplicon.
a. Mix the digestion products from step 2 with 10 mL of 63 DNA loading dye. b. Prepare 1% (wt/vol) agarose gel with Tris acetate-EDTA (TAE) buffer. c. Run the gel at 100 volts (V) for 45 min. d. Cut the desired band from the agarose gel, and dissolve it in one volume binding buffer (XP2) for 7 min at 60 C. e. Run the solubilized gel through a HiBind DNA Mini Column from the Omega Gel Extraction Kit according to the manufacturer's instructions, and elute DNA using 20 mL of water. f. Store the purified digestion products (Figures 2A and 2B) at À20 C for one week.

Ligation.
a. Ligate the enzyme-treated PCR product and pFastBac1 vector from step 3. b. Set up an insert-to-vector molar ratio of 3:1. Master mix can be prepared according to the table in the materials and equipment section. c. Incubate the reaction at 16 C for 12-16 h. 5. Transformation.
a. Thaw 100 mL of Trelief 5a Chemically Competent Cell on ice, and mix them gently with 10 mL of the ligation reaction product from step 4. b. Keep the cells on ice for 15 min.

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c. Heat-shock the mixture at 42 C for 90 s using a water bath followed by incubation on ice for 3 min. d. Transfer the cell suspension to a culture tube containing 0.5 mL of LB medium. e. Incubate the cells with shaking at 200 g for 1 h at 37 C. f. Spread 200 mL of the bacterial cell culture on an LB agar plates with 50 mg/mL kanamycin and incubate the plates at 37 C for 12-16 h. 6. Bacterial cultivation and DNA purification.
a. Prepare growth medium (LB medium containing 50 mg/mL kanamycin). Add 5 mL of growth medium in 14 mL round bottom test tubes. b. Inoculate each tube with a single colony from a successful transformation. c. Grow bacterial culture for 12-16 h in a 37 C incubator with a shaking platform set to 225 rpm. d. Collect the bacteria by centrifugation at 5000 g for 10 min. e. Extract the plasmid harboring the gene of interest using the Qiagen plasmid miniprep kit according to the manufacturer's instructions, and confirm plasmid identity by DNA sequencing. f. Store at À20 C for long-term use.

Protein expression
Timing: 15 days (for step 7) This section describes the expression of recombinant CPP-BoNTA proteins. The expression process in insect cells relies on BEVS and consists of 5 sequential steps: transposition, bacmid isolation, transfection of bacmid into Sf9 cells, viral amplification and protein expression.

Transposition.
a. Prepare LB agar plates containing 50 mg/mL kanamycin, 7 mg/mL gentamicin, 10 mg/mL tetracycline, 100-300 mg/mL Blue-gal and 40 mg/mL IPTG. b. Thaw the DH10BacY competent cells on ice. c. Dispense 100 mL of the cells into 1.5 mL tubes. d. Add approximately 1 mg recombinant donor plasmid (in 5 mL ddH 2 O) and gently mix the DNA into the cells by tapping the side of the tube. e. Incubate the mixture on ice for 30 min. f. Heat shock the mixture at 42 C water bath for 45 s. g. Chill the mixture on ice for 2 min. h. Add 500 mL LB to the mixture. i. Place the mixture in a shaking incubator at 37 C with a shaking speed of 225 rpm for 4 h. j. Dilute 20 mL transposition mixture with 100 mL LB. k. Place 120 mL of each dilution on the plates and spread evenly over the surface. l. Incubate for 24-48 h at 37 C.
Note: Blue colonies may not be discernible at the first 24 h due to the small size. m. Pick white single colony and inoculate into 5 mL LB medium supplemented with 50 mg/mL kanamycin, 7 mg/mL gentamicin and 10 mg/mL tetracycline in 14 mL round bottom test tubes. n. Grow at 37 C to stationary phase (16-24 h) with shaking at 250-300 rpm. 8 e. After centrifugation, transfer the supernatant ($800 mL) to a transparent 1.5 mL tube by careful pipetting. f. Spin the supernatant at 16,000 g for 3 min at 4 C.
Note: This step helps to remove the remaining cell debris, which might decrease the transfection efficiency.
g. After centrifugation, transfer the supernatant ($720 mL) to a transparent 1.5 mL tube by careful pipetting. Add 500 mL isopropanol and thoroughly mix the solution. h. Spin the mixture for 10 min at 16,000 g at 4 C.  Note: The two bacmid preparations from two separate single colonies as described in step 8 are used in transfection to ensure the success of BV production. Each bacmid has two transfection replicates and a non-transfected group is also included. This corresponds to five transfection group on the plate, including bacmid1, bacmid1' (replicate), bacmid2, bacmid2' (replicate) and a control group.
CRITICAL: The final medium volume after transfection should be 2.5-3.0 mL.
e. After transfection, swirl the 6-well plate gently to ensure even distribution of the cells. f. Incubate the transfected 6-well plates at 27 C in the dark. Check the cells every day for contamination and viability.
Note: Successfully transfected cells typically exhibit enlarged size in morphology. The majority of the cells will be infected at the third day post transfection.
g. Collect the medium supernatant at 72 h after incubation. h. Centrifuge the medium supernatant at 1,000 g for 10 min at 4 C to remove cell debris.
i. Harvest the supernatant as initial virus (P1). Store at 4 C in dark for immediate use or at À20 C in dark for long-term use.
Note: Baculovirus is regarded as biosafety level 1 (BSL-1) reagent by authority. However, due to the replicative nature of baculovirus in insect cells, it is preferred to handle baculovirus in virus-specialized cabinet to prevent potential cross contamination.
10. Viral amplification. a. Seed insect cells with viability of more than 95% into 125 mL shake flasks containing Sf-900 II serum-free medium at a density of 1-1.5 3 10 6 /mL. b. Add 2 mL P1 viral stock to suspension Sf9 cells. c. Incubate the cells in a 27 C incubator with a stirring rate of 120 rpm. d. At 72 h post-infection, count and record cell density and viability. e. Collect the medium containing virus (P2) from shaker flasks. f. Centrifuge the virus-containing medium at 500 g for 5 min at 4 C to remove cells and large debris. Store at 4 C in dark for immediate use or at À20 C in dark for long-term use. g. Seed insect cells with viability of more than 95% into 250 mL shake flasks containing Sf-900 II serum-free medium at a density of 1-1.5 3 10 6 cells/mL. h. Add P2 viral stock (1% v/v) to suspension Sf9 cells. i. Incubate the cells in a 27 C incubator with a stirring rate of 120 rpm. j. At 72 h post-infection, count and record cell density and viability. k. Collect the medium containing virus (P3) from shaker flasks. l. Centrifuge the virus-containing medium at 500 g for 5 min at 4 C to remove cells and large debris. Store the supernatant at 4 C in dark for immediate use or at À20 C in dark for longterm use. 11. Protein expression.
a. Seed Hi-5 insect cells with viability of more than 95% into 1 L shake flask containing 300 mL ESf 921 Insect Cell Culture Medium at a density of 2 3 10 6 cells/mL. b. Add P3 viral stock (1% v/v) to suspension Hi-5 cells. c. Incubate the cells in a 27 C incubator with a stirring rate of 120 rpm. d. At 48 h post infection, count and record cell density and viability. e. Collect the cells by centrifuging the culture at 500 g for 10 min at 4 C to remove supernatant. f. Store the cell pellet at À80 C for one year. Pause point: The pellet can be stored at À80 C for one year but prolonged storage is not preferred for purification. Pause point: The protein samples can be stored at À80 C for one year but the activity should be carefully checked after prolonged storage.

Timing: 4 days
Before conducting analysis of the activity of CPP-BoNTA, the integrity and purity of the proteins should be evaluated. This section includes the procedures for three major steps: SDS-PAGE analyses, mass spectrometry analyses and in vitro cleavage assay. A representative result is shown in Figure 3.

Analysis of purity by SDS-PAGE
Note: This step is mainly used for analysis of the purity of the CPP-BoNTA proteins.

Analyses of molecular weight by mass spectrometry
Timing: 2 days In vitro cleavage assay using SNAPtide Timing: 1 day Note: In the SNAPtide, the N-terminal fluorophore is fluorescein isothiocyanate (FITC) and C-terminal quencher is 4-((4-(dimethylamino) phenyl) azo) benzoic acid (DABCYL). Once cleaved, the peptide will release the fluorophore FITC, generating fluorescence signal that can be measured spectroscopically.
16. Evaluation of the in vitro activity of CPP-BoNTA proteins ( Figure 5). In vitro analysis of the cell-penetrating activity of CPP-BoNTA in cell culture

Timing: 3 days
This section describes the procedures for analyzing the cell-penetrating activity of CPP-BoNTA in cell culture. Two major steps are involved including confocal imaging analysis and TissueFax cytometry-like analysis.

Analysis of treated N2a cells by confocal imaging
Timing: 2 days (for step 17) This step describes how to analyze the cell-penetrating activity of CPP-BoNTA by immunofluorescence staining and confocal imaging in mouse neuroblastoma N2a cells ( Figure 6).
17. Mouse neuroblastoma N2a cells culture. a. Maintain N2a cells in MEM EARLES supplemented with 10% FBS, 1% MEM NEAA and 100 U/ mL penicillin/streptomycin at 37 C in fully humidified atmosphere with 5% CO 2 .    (key resources table) with PBS supplemented with 0.2% BSA and incubate with Alexa568-conjugated donkey anti-rabbit IgG (Life/Invitrogen) and Alexa488-conjugated donkey anti-goat IgG (Life/ Invitrogen) secondary antibodies. b. Cover plate at 25 C for 45 min during incubation. c. Wash the coverslips 5 times with PBS supplemented with 0.2% BSA. d. Wash the coverslips 5 times with PBS. e. Wash the coverslips 5 times with PBS supplemented with 0.2% BSA.
Note: From this point on, the slices need to be kept in the dark. The slices can be stored at À80 C for three month.
a. Stain antibody-labeled cells sections with Hoechst 33342 (Invitrogen) for nucleus visualization. b. Cover plate at 25 C for 5-10 min during incubation. c. Wash the coverslips 3 times with PBS. 23. Mounting.
a. Prepare microscope slides and add the coverslips on microscope slides with Prolong gold antifade reagent. b. Leave the microscope slides at 25 C to dry in the dark for 12-16 h. c. Store at 4 C for long-term use. 24. Confocal microscopy imaging.
a. Visualize samples on a LSM710 laser scanning confocal microscopy (Carl Zeiss Microscopy GmbH, Jena, Germany) with the excitation/emission filters for red and green channels to be 493 nm/598 nm and 410 nm/507 nm respectively. 25. Measure the fluorescence intensity in each cell using ZEN 2011 imaging software (Zeiss).
Note: Image analysis is carried out using the program FIJI. Background signal is subtracted from images using the Math function on the FIJI software.

TissueFAXS cytometry analysis
Timing: 1 day (for step 26) This step describes the procedures for flow cytometry-like epifluorescence analysis using The TissueFAXS system. This approach is useful for quantification of a large set of samples.
26. Visualize the prepared samples from step 23 on TissueFAXS (TissueGnostics, Vienna, Austria) fluorescence imaging system. Scan the whole section slices and calculate the fluorescence intensity using TissueQuest software (Figure 7).
In vivo analysis of the efficacy and cell-penetrating activity of CPP-BoNTA in mice

Timing: 7 days
This section describes the procedures for the in vivo analyses of CPP-BoNTA in mice. Two major steps are included: analysis of the efficacy of CPP-BoNTA using digit abduction score, and analysis the cell-penetrating in gastrocnemius muscle.

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In this step, we use digit abduction score (DAS) assay, which measures muscle-weakening effectiveness, 9 to determine the pharmacologic activity (ED 50 ) of BoNTA proteins. Therapeutic index is calculated using the determined ED 50 and systemic toxicity (LD 50 ).
27. Injection procedure ( Figure 8). a. Prepare 8 to 12-week-old male or female C57BL/6 mice for intramuscular injection. For each experiment, inject at least five mice per dose (Table 1). b. Dispense different doses of CPP-BoNTA according to the body weight of the mice, so that the mice of each doses receive a fixed volume of 5 mL. c. Attach a 30-gauge needle to a sterile 250 mL Hamilton syringe. d. Inject CPP-BoNTA or vehicle (0.9% saline) into the head of the right gastrocnemius muscle of mice. Note: It is preferred to include 5 or more mice for each dose to make the results more reliable.
CRITICAL: Due to the small hind limbs of mice, it is necessary to avoid the needle to pass through the gastrocnemius muscle, which will result in ineffective injection.
CRITICAL: It is necessary to avoid bleeding during the injection, otherwise the mice may lick the wounds and swallow the injected proteins. If bleeding is caused, push the wound with an alcohol cotton ball to stop the bleeding and then wipe off the blood.  Table 1.
CRITICAL: Two observers are required for DAS measurements to ensure that the data obtained are unbiased. The two observers must be blinded to the treatment, and at no time should they communicate with regard to the DAS values that they assign.
Note: Data are shown as mean G standard deviation (SD).

Determination of the systemic effects and therapeutic index.
a. Determine the half intramuscular lethal dose (IMLD 50 ).
Note: IMLD 50 is defined as the dose at which half of the mice die following treatment. Set the end point of monitoring at day 5, after which no further death can be found ( Figure 10A).

EXPECTED OUTCOMES
BoNTA is widely used in treating neuromuscular disorders. Commercial BoNTA drugs are prepared by fermentation of Clostridium botulinum. Here we provide a step-by-step protocol for producing recombinant BoNTA proteins harboring cell-penetrating peptides using the BEVS-insect cell expression system. Following this protocol, highly homogenous proteins can be obtained ( Figure 3). These proteins exhibited improved therapeutic index ( Figure 10) and enhanced cellular uptake (Figure 11) in mice.

LIMITATIONS
The in vivo activity of purified BoNTA proteins is determined using digit abduction score. 9 While this method is the gold standard for academic and industrial applications, it is a semi-quantitative method that relies on the visual inspection from two researchers. This may generate variations under different experimental settings, such different researchers. Therefore, cautions should be taken when the results are interpreted. In addition, this protocol should be only used for academic scale protein production.

Problem 1
The concentration of isolated and purified bacmids are too low (step 8: Bacmid preparation).

Potential solution
In the process of bacmid isolation, avoid the DNA fragmentation due to shearing force. In addition, pre-chilled isopropanol and 70% ethanol can increase the efficiency of DNA precipitation. Last, it is proper to leave some residual isopropanol or ethanol to avoid the disruption and loss of DNA ll OPEN ACCESS sample given sufficient time is provided to air dry the residual isopropanol or ethanol. Generally, the concentration of purified bacmids should be more than 1,000 ng/mL.

Potential solution
Make sure sterile environment is maintained when removing 70% ethanol from the tube.

Problem 3
During the purification step, a large amount of target protein remains in the cell pellet (step 12: Purification with Ni-NTA agarose beads).

Potential solution
Increase the concentration of salt ions in the lysis buffer. We find that a solution containing 2 M NaCl can help improve the yield and purity of CPP-BoNTA proteins. Figure 11. Evaluation of the in vivo cellular uptake of intramuscularly injected CPP-BoNTA proteins in mouse gastrocnemius muscles using immunofluorescence staining Scale bar, 200 mm. The data in the bar plot are presented as mean G SD. The significant differences between BoNTA and T-BoNTA or Z-BoNTA are analyzed using Student's t test. The p values are indicated.

Problem 4
When N2a cells are visualized by confocal microscopy, many cells are overlapped, making it difficult to observe fluorescence (step 17: Mouse neuroblastoma N2a cells culture).

Potential solution
When the cell density is too high, N2a cells tend to aggregate rather than uniformly distributing in the coverslips. Therefore, attention should be paid during cell seeding process.

Problem 5
During the immunofluorescence experiment, the fluorescence signals of the two channels interfere with each other, resulting in the confusion of the received fluorescence signals (steps 24 and 36: Confocal microscopy imaging).

Potential solution
Fluorophore-conjugated secondary antibody used in different channels should be carefully selected to avoid overlap in the emission filters. When receiving the fluorescence signal with small emission filters, attention should be paid to adjust the range of the filters.

Problem 6
During the DAS experiments, high frequency of injection failure or body injury are observed in mice due to improper experimental manipulations (step 27: Injection procedure).

Potential solution
Hold the mouse by the tail and let it cling to the cage, so that its whole body is in a stretch state. And then press the mouse's back with the palm of the hands while slowly moving your fingers from the back towards the neck with thumb and index finger. This can grasp the skin on the back of the mouse neck and thus calm down the mice for further experimentation.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jia Liu (liujia@shanghaitech.edu.cn (J.L.).

Materials availability
Plasmids, primers, recombinant proteins, and any other research reagents generated by the authors will be distributed upon request to other research investigators under a material transfer agreement.

Data and code availability
This study did not generate or analyze datasets or code. The published article includes the figures generated with this protocol.